A Thesis Submitted to the Faculty of Graduate Studies and Research In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology University of Regina

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dc.description.abstract

The cell envelope of Gram-negative bacteria is a complex structure that is critical
for facilitating interactions between bacteria and their environment. The objective of this
research was to identify and characterize novel genes required for proper cell envelope
development in the legume symbiont Rhizobium leguminosarum.
In R. leguminosarum there is a positive correlation between mutations that affect
cell envelope structure and sensitivity to peptide-rich media; therefore, I implemented an
innovative transposon mutagenesis approach to identify novel genes connected to the cell
envelope by screening for mutants unable to grow on peptide-rich media, but unaffected
in growth on minimal media. Of the 10 mutants isolated, 5 of the mutations were mapped
to cell envelope components; therefore, the genetic screen was successful. Three mutants
were chosen for further study because the mutations were in uncharacterized genes that
affected the cell envelope function.
One of the mutations was mapped to the putative ATP binding component of an
ABC transporter. This mutant had significantly decreased levels of exopolysaccharide
(EPS) compared to wild-type, and was also desiccation sensitive and unable to form
biofilms. Another mutation mapped to the gene fabF2XL, which is required for synthesis
of the unique lipid A very long chain fatty acid (VLCFA). The third mutation mapped to
a conserved hypothetical membrane protein, RL3501, that is part of a four gene operon.
Both of these genes were critical for maintaining membrane stability in the presence of
detergents, hydrophobic antibiotics, and antimicrobial peptides. Mutation of fabF2XL or
RL3501 also had a negative impact on plant infection, and expression of an outer membrane protein gene, ropB, indicating that they are crucial for proper cell envelope
development in free-living conditions and during symbiosis. I also identified novel
phenotypes associated with mutation of the sensor kinase chvG that regulates cell
envelope structure. Collectively, the results of this study have identified new cell
envelope genes that are critical for proper envelope function, and contributed to the
development of a new model for cell envelope development in Rhizobium
leguminosarum by revealing novel mechanisms for the regulation and interaction of key
envelope components.

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dc.description.uri

A Thesis Submitted to the Faculty of Graduate Studies and Research In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy *, University of Regina. *, * p.